Structural basis of RNA polymerase II transcription on the chromatosome containing linker histone H1

In chromatin, linker histone H1 binds to nucleosomes, forming chromatosomes, and changes the transcription status. However, the mechanism by which RNA polymerase II (RNAPII) transcribes the DNA in the chromatosome has remained enigmatic. Here we report the cryo-electron microscopy (cryo-EM) structures of transcribing RNAPII-chromatosome complexes (forms I and II), in which RNAPII is paused at the entry linker DNA region of the chromatosome due to H1 binding. In the form I complex, the H1 bound to the nucleosome restricts the linker DNA orientation, and the exit linker DNA is captured by the RNAPII DNA binding cleft. In the form II complex, the RNAPII progresses a few bases ahead by releasing the exit linker DNA from the RNAPII cleft, and directly clashes with the H1 bound to the nucleosome. The transcription elongation factor Spt4/5 masks the RNAPII DNA binding region, and drastically reduces the H1-mediated RNAPII pausing.


REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In this manuscript, Rina Hirano et al. determined the Cryo-EM structures of transcribing Pol II-chromatosome complexes in two forms. The two structures provide the mechanism of H1 pauses Pol II transcript and show the sequential process of Pol II transcript on the chromatosome. While the results are interesting, the manuscript was not well prepared and should be largely improved before publication.

Reply)
Thank you very much for your favorable evaluation. We have revised the manuscript according to your suggestions.
Major concerns: 1. The H1 is commonly thought to compact chromatin and repress transcription.
Although the H1 function is critical for transcriptional regulation, a direct connection between H1-containing nucleosome and Pol II elongation complex remains controversial.
The authors should discuss previous finding and clarify this issue in their manuscript to avoid misleading to readers.

Reply)
Thank you very much for this comment. In the revised manuscript, we explained that most H1 subtypes function as transcription suppressors in the Introduction, and added previously obtained information about H1.2 as a transcription suppressor and activator with new citations (p. 3, ll.55-57 and p.4, ll.66-76). These additional explanations will avoid misleading the readers.
2. The authors should fit their structures and working model into nucleosome array, which represent the natural chromatin with evenly-spaced nucleosomes (linker DNA ~20 to 30 bp). For example, there is no exposed end in cells when Pol II elongation complex proceeds on chromatin (with H1 or not). In the complex structure in form-1, the exit DNA generates clash with Pol II. From the figure, I can't see the clash in detail. The authors may describe what the structure/complex would be if the exit DNA has no exposed end and if the exit end associates with a nucleosome. This illustration will clarify whether this conformation fits the complex in the physiological context.

Reply)
Thanks again for this comment. In the revised manuscript, we illustrated an RNAPII 3. Fig. 1c shows that there are nearly half of Pol II transcribed into the nucleosome in the presence of H1. The result seems to be inconsistent with the structural observation and the conclusion of the manuscript. Did the author observe particles showing that Pol II enters into the nucleosome?

Reply)
As this reviewer pointed out, the RNAPII transcribed into the nucleosome in the presence of H1. We described this fact in the text (p. 5, ll.104-106). In addition, we have tried to capture the RNAPII-chromatosome particles, in which RNAPII enters into the nucleosome. However, we could not detect such particles in our cryo-EM images. This should be an interesting project in the future.
4. The figures, main text (including the discussion) were not well prepared and should be improved before publication.

Reply)
In the revised manuscript, we extensively revised the figures and text according to this reviewer's suggestions. Please find these changes in the revised text and figures. These changes are explained in our responses to each reviewer's comments.
Minor concerns: 1. Fig. 2a, Fig.3a, and 3b are not very clear. The authors should consider changing the model color, or the model display.

Reply)
In the revised manuscript, the colors of the figures have been changed to make them easier to understand for people with color blindness.
2. The assembly of RNAPII-chromatosome complexes. Why not assembled the chromatosome before loading the pol II on the template nucleosome?

Reply)
We added the H1 after the RNAPII loading, because the H1 bound to the bubble DNA region and inhibited the RNAPII loading. We described this fact in the revised manuscript (p.5, ll.100-101).
3. The authors should show the SDS-PAGE of purified proteins, and angular distributions and local resolution estimation of the cryo-EM reconstructions.

Reply)
We presented the SDS-PAGE gels of purified proteins, and the angular distributions and Some issues need to be clarified.

Reply)
Thank you very much for your favorable comments. According to this reviewer's suggestions, we revised the manuscript as outlined below.
1. The authors used yeast Komagataella pastoris (belongs to the family of budding yeast) RNAPII and human linker histone. Budding yeast chromatin has much shorter linker DNA and no canonical H1. I suppose the authors are trying to mimic high-order systems in this study. If so, they should test if a similar reaction product is produced using mammalian RNAPII. At least the authors should discuss the rationale for choosing this hybrid system, for example, by comparing the structures of mammalian and yeast RNAPII.

Reply)
As pointed out by this reviewer, it is important to test a mammalian RNAPII in the current chromatosome transcription system. Therefore, in the revised manuscript, we prepared mammalian RNAPII from Sus scrofa domesticus, and performed the chromatosome transcription assay. Like the K. pastoris RNAPII, the S. scrofa RNAPII exhibited similar pausing on the entry linker DNA in the H1.2-dependent manner. Therefore, we concluded that the K. pastoris RNAPII pausing mechanism in the chromatosome may be conserved in mammalian RNAPII. These new data are presented in the new Fig. 1e, and the results are described in the text (p.6, ll.119-129).
2. Two bands at ~60-nt exist in the absence of H1, suggesting that the product is not determined by the specific interactions between RNAPII and H1. It needs to be pointed out.

Reply)
Thank you very much for this comment. As this reviewer pointed out, the band around 60-nt was weakly observed in the absence of H1. This suggests that the exit linker DNA may be trapped by the processing RNAPII with its DNA-binding cleft in the absence of H1, but its efficiency is low because of the flexibility of the exit linker DNA. This is consistent with the idea that H1 binding restricts the linker DNA flexibility, and enhances the RNAPII pausing rate. We discussed this point in the Discussion section of the revised manuscript (p.10, l.209-p.11, l.219).
3. The structure form I shows that the linker DNA blocks the RNAPII. It is hypothesized this is the mechanism for the inhibition. The two DNA linkers is held together by linker histone tails. It would be important to verify the hypothesis by studying the effects of removing the H1 C-terminal domain on transcription.

Reply)
Thank you very much again for this comment. As this reviewer suggested, we performed the chromatosome transcription experiments with the H1.2 mutant, in which the Cterminal DNA binding region (amino acid residues 151-212) was deleted. We then found that the RNAPII pausing on the entry linker DNA was still observed, with a slightly reduced rate as compared to the full length H1. Therefore, the C-terminal DNA binding region of H1 partly contributes in the RNAPII pausing. These new data are presented in the new Fig. 1d, and the results are described in the text (p.6, ll.111-118).

Related to 3, this study would benefit from the inclusion of RNAPII elongation studies
performed with di-nucleosomes.

Reply)
According to this reviewer's suggestion, we performed the chromatosome transcription assay with di-nucleosomes, in which two nucleosomes are connected with 48 base pairs of linker DNA. The 48 base-pair linker DNA is known as an average linker length in transcriptionally active loci in human cells. We then found that the RNAPII pausing on the entry linker DNA of the upstream nucleosome was substantially enhanced by the H1 binding. Therefore, we conclude that the RNAPII pausing found in the present study may occur in the natural context of chromatin. These new results are presented in the new 5. If the authors believe the basic patch is critical for pausing, mutations that remove the basic patch should be made to test their hypothesis (if the experiment is doable.)

Reply)
To test the functional importance of the RNAPII basic patch, we performed a chromatosome transcription assay with the transcription elongation factor Spt4/5, which is known to mask the RNAPII basic patch. We obtained the important finding that Spt4/5 drastically alleviates the entry barrier by the chromatosome formation, probably by interfering with the binding of the exit linker DNA on the RNAPII surface. This new finding provides additional evidence that the RNAPII basic patch functions to induce the H1mediated RNAPII pausing. In the revised manuscript, we added these new data in the new Fig. 5, and discussed the in the new Results section "The transcription elongation factor, Spt4/5, alleviates the H1-mediated RNAPII pausing" (p.9, l.185-p.10, l.200).
It would also be interesting to test RNAPII mutants with basic patch mutations or deletions. However, as this reviewer noted, we need to establish a new expression system for the RNAPII mutants and their purification schemes. Therefore, it will take a long time to complete the mutational studies for RNAPII. Accordingly, we think this is an excellent project for future studies.